Liquid phase columnar separations are among the workhorse techniques for a present day analytical chemist. Capillary electrophoresis (CE) and especially high performance liquid chromatography (HPLC) are the two dominant examples. In either one, the sample is injected at one end of a tubular conduit and moved towards the exit of the conduit either by a pumped eluent flowing through the conduit (e.g., HPLC) or by applying an electrical field applied across the length of the tube (e.g., CE). Towards the end of the conduit (CE, capillary HPLC) or immediately after the conduit a detector is located. Separation of different constituents in the sample is achieved because they move at different rates through the column and arrive at the detector at different times.
Particularly in HPLC to achieve time-efficient separation and sensitive detection, a technique called gradient elution is used, especially when the sample contains both (a group of) weakly retained (fast moving) species and (a group of) strongly retained species. In this case, a weak eluent (low pushing power) is used during the initial phase of the separation so that separation is achieved between the different weakly retained species. Then the eluent strength is increased to elute the strongly held species and achieve separation between them. If the weak eluent is used throughout, the strongly held analytes will take a very long time to elute (if they elute at all) and the resulting peaks will be very broad, making sensitive detection difficult. If the strong eluent is used throughout, the weakly retained species will elute together as a group, undifferentiated.
There are many programs that simulate separations described above, including one recently developed by the inventors. See Reference (23) below. Such simulations and real life experiments indicate that even in isocratic (i.e., not involving a gradient), the separation of the analytes is often complete long before it reaches the terminal detector, not only time is wasted but the bands broaden over the rest of the time and detection sensitivity is decreased. In gradient elution, it is not uncommon that if the strong eluent concentration is increased too soon, a pair of weakly retained species that were separated as they resided on the column ultimately got pushed into one undifferentiated peak. Picking a particular temporal gradient program is rarely approached a priori, rather one tries to fine tune this as a result of several successive trials of varying composition.
Ironically when Mikhail Tswett first invented chromatography, he separated plant pigments on a glass column filled with CaCO3 and he was able to observe his separation visually. Although admittedly he did not have any means in his time to quantitate the bands on the column. In principle, if you can not only see the separation in a qualitative manner but in a quantitative fashion with good sensitivity, there is no need to wait until an end-column detector sees the eluted band. Indeed, if one can see the separation in near real time, one can alter the elution conditions concurrently to achieve the best separation and the most sensitive quantitation possible.
The concept of quantitatively detecting/imaging what is present in different locations in the column at all times during the separation process is not new. The term “Whole Column Detection” (WCD) was coined first to the inventors' knowledge by Birks and his students in a theoretical simulation. See Reference (1) below. In traditional chromatography with a terminal post-separation detection arrangement, the only identifying marker of an analyte independent of any specific detector is the terminal retention time, often described in terms of its “retention factor”. Note that spectral characteristics and such others that are sometimes used for identification are not intrinsic to chromatography, that information is detector specific.
Obviously to perform whole column detection, the detector must be able to “see through” the bounding walls of the column. As early as 1968, Brumbaugh and Ackers described “scanning gel chromatography” where the column was moved past a fixed light source-detector configuration and were able to monitor the absorbance profile of molecular sieve separations throughout the length of a gel column. See Reference (2) below. Previously, moving a HPLC column (to which today pumps and an injector are integrally attached) is not very practical. In their first experimental paper, Birks and his students used a metal jacketed glass column, a long fluorescent lamp emitting at 365 nm on one sides and 14 pairs of holes opposite each other in the metal jacket: light entered on one side and was detected with 14 individual photodiodes on the other side. See Reference (3) below.
While a number of the theoretical predictions and an elucidation of how retention factors change under gradient conditions could be verified and demonstrated, with a maximum resolution of 14 points along the column, performance and accuracy of the results were less than desirable.
Pawliszyn used his considerable prior experience in detecting refractive index changes in a capillary to design a refractive index gradient detection system over an effective length of 15 mm in a capillary isoelectric focusing (CIEF) analysis system. See Reference (4) below. A stationary focused He—Ne laser source was used. The beam was expanded by a lens after the capillary such that the distance on the capillary was magnified 10 times in the detector plane where the scanned distance was 150 mm with a resolution of 0.1 mm (a syringe pump drive was adapted) providing a 1500 point resolution over the entire separation distance of 15 mm. Because it was not possible to move the single detector photodiode at sufficient speeds needed for fast electrophoretic separations, they also imaged a smaller (3 mm) length of the separation capillary with a 128 element photodiode array. A wider photodiode array would have allowed a longer portion of the separation capillary to be directly imaged and the moving photodetector can be dispensed with. Precisely this was done in 1994 with an argon ion laser source and a 1024—element CCD detector—the 25 mm wide detector imaged 25 mm of the capillary (with the resolution obviously being 1024 points). See Reference (5) below; see also U.S. Pat. No. 5,395,502.
The same concept as above was adapted by Beale and Sudmeier in 1995 to CE or CIEF with laser-induced fluorescence (LIF) detection. See Reference (6) below. They placed the entire separation capillary on a motorized translation stage (max speed 50 mm/s), up to 19 cm length could be brought under the field of view of a microscope objective. The LIF system operated in the confocal mode. They obtained better results with conventional capillaries from which the polyimide coating was removed (with either fuming sulfuric acid or a butane torch) than a silica capillary with UV-transparent coating. They noted that with inner bores <75 μm, it becomes very difficult to maintain laser focus inside the capillary, which greatly decreases signal-to-noise (S/N).
It is also to be noted that few substances have native fluorescence. Any analysis system relying on LIF must undergo prior derivatization. No suitable derivatization chemistry may exist; at the very least this represents an extra cumbersome step. Photobleaching of fluorescence with repeated scanning is also a problem, especially with an intense source.
In 1996, Preisler and Yeung illuminated 232 mm of a capillary with an Ar laser and a plano-convex lens. See Reference (7) below. The entire illuminated area was monitored through a perpendicularly mounted 578 element CCD array equipped with an appropriate emission filter.
They merely monitored the movement of a fluorescein band/front to determine flow velocity but in principle this will allow monitoring the separation of analytes that can be made to fluoresce with the particular laser source with the caveats already outlined. Prior to this in 1994 an arrangement was demonstrated by Wu and Pawliszyn where the input light was coupled by a fiber-optic array to the capillary but the sensitivity was poor as the light coupling was not efficient. See Reference (4) below.
In 1998 based on Pawilszyn's work, Convergent Biosciences in Canada commercialized an imaging CIEF detection system that uses a fiber optic array to bring in 280 nm UV light from a Xenon lamp into a capillary cassette with a 50 μm wide 5 cm long aperture.
The transmitted light is read by a CCD array. This instrument (iCE280) is still sold as such and as part of a more elaborate iCE3 system.
In 2001 in their review of imaging detection in CE and CIEF, Wu et al. summarized the status of the field at that time. See Reference (8) below. The favored generic arrangement of illumination and detection, whether by transmittance or fluorescence, is shown in the article; the iCE280 arrangement does fall in this category.
It is believed that the iCE280 is thus far the only commercial instrument to offer whole column or imaging detection and it is ideally suited only when the total separation distance is small, e.g., 5 cm for the iCE280. This is applicable in CIEF but there are few other techniques where this can be accepted.
A wholly different approach is possible with a liquid core waveguide (LCW) both for absorbance and fluorescence detection. An LCW is a tube or conduit where the wall is composed of a material that is both optically transparent in the wavelength region of interest.
Light can proceed through a long LCW capillary with relatively small loss. If such a capillary is axially illuminated and the light passing through the entire length of the capillary is monitored, as soon as the sample is injected light transmission goes down due to the light absorbing components present in the sample. The signal will remain unaltered until the earliest eluting component falls off the detection path—the transmitted light will rise by that amount. If this data is depicted as absorbances vs. time, the output will resemble a downward stair case with the transition from each step to another depicting the elution of an analyte. A more conventional chromatogram or electropherogram can be obtained by differentiating the signal with time.
Although this concept was demonstrated with regular capillaries (which lose a lot more light, see Reference 9 below), the process becomes more practical with a LCW capillary (see Reference 10 below). Nevertheless, this system has numerous difficulties. Even though a large absorbing peak may be completely separated, they appear in the signal together. If the small one elutes first its quantitation accuracy is limited by the need to subtract one large number from another. Differentiation magnifies noise. Much of the time detection is done with a number of absorbing components in the light path this reduces light throughput and increases detector noise.
Fluorescence detection with a liquid core waveguide tube has more possibilities. If the excitation light is radially incident on the capillary, the unabsorbed incident radiation largely passes out through the wall. In contrast, a significant portion of the emitted fluorescent light proceeds down the tube where it can be picked up either by a fiber optic coupled to a photodetector or directly by a photodetector. See References (11) and (12) below.
Instead of trying to illuminate the tube uniformly along its length, a laser beam can be scanned (either through space or coupled by a fiber optic) along the separation capillary, revealing where the fluorescently labeled analytes are located. In 2002, Olivares et al. described such a system and used it for both CE and CIEF over a scanning length of 12 cm. See Reference (13) below.
There are some complications with such an arrangement, aside from the general problems with fluorescence detection already mentioned. Except when the analyte is the nearest one the detector, any fluorescence elicited and traveling to the detector must travel through other analyte band(s) between it and the detector and light will be lost by absorption making quantitation complicated.
The roles of the axial and radial light can be reversed. The LCW can be illuminated axially and the fluorescence radiation exiting the wall can be read by an imaging detector/camera. See Reference (14) below. Other applications of this configuration were discussed by the senior author in a 2005 review. See Reference (15) below. However, this configuration has even more problems than the one just discussed from axial light loss due to absorption by preceding analyte zones and accurate quantitation is difficult. This was described in U.S. Pat. No. 6,852,206 but for reasons above, never commercialized.
An approach that is similar to Wu and Pawliszyn's 1992 paper (see Reference (4) below) in that the column was uniformly illuminated along its length and the detector (in this case a CCD array, rather than a photodiode), registering a portion of the column was moved along (in this case by an optical scanner drive, rather than a syringe pump drive) was described by Lin et al in 2008. See Reference (16) below. They used however not an open tubular capillary but a 3 mm ID glass tube with 10 μm octadecylsilane bonded silica particles. This was then inserted into a stainless steel tube with windows on opposite sides cut in it. The authors stated that the system permitted a resolution of 0.3 mm. As with the apparatus described in Reference (4), light coupling in and out of the column was through space.
It is important to note that that during gradient elution the analyte does not move at a constant speed throughout: the entire journey of an analyte—the two dimensional space-time transit map of the analyte, as it were—can serve as a much more specific and discriminating marker rather than a one dimensional specification of when a given analyte “finished the race”.
Currently, detection for chromatography is done in a fixed position, typically after elution from the column. This means that not only must one wait for a period of time for all of the analytes to elute, but also that the time used to perform the separation is inefficient. This is due to the fact that, though a separation may be complete in the first 10% of the column, it is unknown until it reaches the detector. Additionally, if a gradient elution method is used, it is possible to have separated analytes before the increase in eluent strength, but have them co-elute when stronger eluents are applied.
What is needed is a system for detecting the elution of analyte along a separation conduit in real time, allowing for more efficient separation and detection of analytes in a given sample.